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  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the invention relates to a method of predicting response to blood pressure (BP) lowering agents .
• the invention relates to a method of predicting a response to angiotensin-2-receptor blocker and renin inhibitor classes of BP lowering agents .
• renin angiotensin aldosterone system RAAS
• RAAS renin angiotensin aldosterone system
• Renin is of considerable importance as the first and rate limiting step in the RAAS system. It was noted as long ago as 1972 that hypertensive patients with high renin levels were more likely than those with normal or low renin levels to experience myocardial infarcts . Synthesis and secretion of renin by kidney juxtaglomerular cells is upregulated by cAMP, (mainly through beta 1 adrenergic receptor agonism) , and down regulated by angiotensin II and intracellular calcium, in response to salt load, renal perfusion pressure, noradrenaline and renal prostaglandins . 5 Several DNA elements in the distal and proximal promoter regions as well as in intron 1 have been implicated in cAMP regulation . A high molecular weight inactive renin is formed when renin combines with its endogenous inhibitor renin binding protein (ReBP) . 6
• ReBP endogenous inhibitor renin binding protein
• Dispite renin being the key enzyme of the RAAS system, and therefore a good candidate quantitative trait locus for elucidating the molecular and genetic influences implicated in the molecular etiology of essential hypertension, a consensus concerning the nature, frequency and functionality of SNPs in the renin gene has not yet been reached .
• a very small sib-pair analysis ( 66 dizygotic twins and their parents) found both diastolic and systolic BP to be linked to the renin gene locus . 8 However two other linkage studies yielded negative results .
• heterozygous or homozygous carriers of the T allele of the (C-5312T) renin single nucleotide polymorphism SNP
• SNP single nucleotide polymorphism
• the invention relates to a method of determining a suitable blood pressure lowering treatment for an individual, comprising a step of assaying a biological sample from the individual for the presence or absence of the C-5312T SNP in a distal enhancer region of the renin gene, wherein the presence of at least one T allele is indicative of an increased response to a blood pressure lowering treatment selected from the group comprising : angiotensin-2-receptor blockers ; ACE Inhibitors ; aldosterone receptor blockers ; and beta-receptor blockers, and wherein the absence of a T allele (i . e homozygous for the C allele) is indicative of an increased response to a blood pressure lowering treatment selected from the group comprising : renin inhibitors ; calcium channel blockers ; and diuretics .
• the presence of the SNP indicates that the individual is likely to respond better to the first group of drugs as compared to the second group of drugs, wherein response is defined as extent of BP lowering achieved.
• the presence of at least one T allele is indicative of an increased response to a blood pressure lowering treatment selected from the group comprising : angiotensin-2-receptor blockers ; ACE Inhibitors ; and aldosterone receptor blockers .
• the presence of at least one T allele is indicative of an increased response to a blood pressure lowering treatment selected from the group comprising : angiotensin-2-receptor blockers ; and ACE Inhibitors .
• the presence of at least one T allele is indicative of an increased response to a angiotensin-2- receptor blocker .
• the absence of the T allele is indicative of an increased response to renin inhibitors .
• the presence of at least one T allele is indicative of an increased response to LOSARTAN .
• the absence of at least one T allele is indicative of an increased response to ALISKIREN .
• both alleles of the rennin gene are assayed for the presence of the SNP .
• Any suitable biological sample which contains genetic material may be used as a biological sample for the assay of the present invention.
• Particularly suitable samples comprise saliva and leucocyte preparations .
• the individual is selected from the group comprising : individuals having established elevated blood pressure; individuals at risk of developing elevated blood pressure; and individuals identified as having elevated cardiovascular risk.
• the method of the invention may also be used with individuals that do not fall into any of the above-mentioned groups .
• the pharmacogenomic assay of the present invention may form a part of a genetic screening assay of individuals that are not at risk of cardiovascular disease, high blood pressure, or hypertension .
• An individual identified as carrying the SNP in at least one allele of the rennin gene is typically subj ected to a heart disease risk factor management programme .
• Individuals found to be homozygous for the T allele will ideally be subj ected to an aggressive heart disease risk factor management programme .
• the term "heart disease risk factor management programme" should be taken to mean at least one of lifestyle management and pharmaceutical intervention, and wherein pharmaceutical intervention ideally includes treatment with at least one of RAAS blockade therapy, statin therapy, and anti-oxidant therapy.
• the invention also relates to an assay kit for use in determining a suitable blood pressure lowering treatment for an individual and comprising:
• a suitable blood pressure lowering agent for the individual wherein the presence of at least one T allele is indicative of an increased response to a blood pressure lowering treatment selected from the group comprising: angiotensin-2-receptor blockers ; ACE Inhibitors ; aldosterone receptor blockers ; and beta-receptor blockers , and wherein the absence of a T allele (i . e . CC homozygotes ) is indicative of an increased response to a blood pressure lowering treatment selected from the group comprising : rennin inhibitors ; calcium channel blockers ; and diuretics .
• the instructions specify that the presence of at least one T allele is indicative of an increased response to a blood pressure lowering treatment selected from the group comprising : angiotensin-2- receptor blockers ; ACE Inhibitors; and aldosterone receptor blockers .
• the instructions may specify that presence of at least one T allele is indicative of an increased response to a blood pressure lowering treatment selected from the group comprising: angiotensin-2-receptor blockers ; and ACE Inhibitors .
• the instructions may specify that the presence of at least one T allele is indicative of an increased response to a angiotensin-2-receptor blocker .
• the instructions specify that the absence of a T allele is indicative of an increased response to renin inhibitors .
• the instructions specify that the presence of at least one T allele is indicative of an increased response to LOSARTAN .
• the instructions specify that the absence of a T allele is indicative of an increased response to ALISKIREN .
• the invention also relates to a method of predicting the likelihood of an individual having an atherothrombotic event, comprising the step of assaying a biological sample from the individual for the presence of a C-5312T single nucleotide polymorphism ( SNP) in at least one allele of the distal enhancer region of the renin gene, wherein the presence of the SNP in at least one allele of the gene indicates that the individual is at an increased risk of having a future atherothrombotic event as compared to an individual homozygous for the C allele .
• SNP single nucleotide polymorphism
• the biological sample from the individual is assayed for the presence of the C-5312T single nucleotide polymorphism ( SNP) in both alleles of the distal enhancer region of the renin gene, wherein the presence of the SNP in both alleles of the gene (TT homozygous) indicates that the individual is at an increased risk of having a future atherothrombotic event as compared to an individual homozygous and/or heterozygous for the C allele .
• SNP C-5312T single nucleotide polymorphism
• the method involves measuring the blood pressure of the individual , wherein the presence of the SNP in both alleles of the gene, combined with elevated blood pressure, indicates that the individual is at an increased risk of having a future atherothrombotic event .
• identified TT homzygotes are subj ected to a heart disease risk factor management programme .
• the invention also relates to an assay kit for use in identifying individuals at an increased risk of having a future atherothrombotic event, comprising:
• the instructions specify that the presence of the SNP in both alleles of the gene (TT homozygous ) indicates that the individual is at an increased risk of having a future atherothrombotic event as compared to an individual homozygous for the C allele .
• the instructions specify that the presence of the SNP in both alleles of the gene (TT homozygous) indicates that the individual is at an increased risk of having a future atherothrombotic event as compared to an individual heterozygous for the T allele .
• the instructions additionally specify measuring the blood pressure of the individual, wherein the presence of the SNP in one, and ideally both, alleles of the gene, combined with elevated blood pressure, indicates that the individual is at an increased risk of having a future atherothrombotic event compared with an individual homozygous or heterozygous for the C allele .
• angiotensin-2-receptor blockers examples include losartan, irbesartan, telmesartan, candesartan .
• ACE inhibitors examples include perindopril, enalopril, ramapril, and lisinopril .
• beta-receptor blockers examples include metoprolol , and atenolol .
• aldosterone receptor blocker is spironolactone .
• calcium channel blockers examples include amlodipine, nifedipine, and felodipine .
• Assay kits for detecting the presence or absence of the renin C-5312T SNP will be well known to those skilled in the art .
• assay kits based on RT PCR will be known, as will suitable primers for use in such reactions .
• Assay kits that are not based on PCR technology are also known in the art .
• the design of probes for use in such kits is also within the ambit of the person skilled in the field of molecular biology.
• WO 95/11995 describes the design and principles of use of arrays of oligonucleotide probes for use in genotyping SNP' s', and in which the existence of a given SNP is manifested by differences in normalised hybridisation intensities of probes flanking the SNP when the probes hybridise to corresponding targets from different individuals .
• Saiki et al 17 describe the design and use of allele-specific probes for analysing SNP' s .
• Genotyping of the renin gene C-5312T polymorphism was performed using the Amplifluor technology 19 by KBioSciences Ltd (www . kbioscience . co . uk) . In brief, genotyping is performed in 384-well microplates using a fluorescence resonance energy transfer ( FRET) -based genotyping method. Amplification was initiated using the following allele-specific primers and a common downstream primer .
• FRET fluorescence resonance energy transfer
• AAAGCAGTCTCTGTAAGTGCCC SEQUENCE ID NO.3
• the allele-specific primers are tailed with unique sequences that create corresponding complementary sequences in the two amplicons .
• quenched Universal AmplifluorTM primers in a hairpin formation
• These primers contain 3' tails that specifically bind to the unique tailed sequences in the amplicons and continue amplification .
• the action of the DNA polymerase opens up the hairpin structure and the quencher and reporter moieties are spatially separated .
• the excited reporter moiety emits either red or green fluorescence, the colour of which depends on which nucleotide is at the polymorphism site .
• the fluorescence is quantified by a microplate reader and then analysed via an Excel macro to provide genotypes for each SNP .
• 20 ⁇ L of total DNA was supplied to the company at 2ng/ ⁇ L ( 3ng is consumed per assay) in "v-bottomed" 96-well micro-titre plates .
• Also included were a number of repeat (83) and blank samples ( 31) to check for reproducibility and to control for errors in sample handling .
• Table 1 shows the -5312 C/T SNP genotype frequencies amongst the hypertensive participants in the clinical trial compared to a community dwelling normal population . Despite similar allele frequencies , in contrast to the control normal population, the genotype distribution amongst the hypertensive population was clearly not in Hardy-Weinberg equilibrium, principally due to a paucity of TT homozygotes, and a small excess of CT heterozygotes . A likely explanation for the paucity of TT homozygotes is their exclusion from participation in the clinical trial . Relevant exclusion criteria for the trial included inability to withdraw from current antihypertensive medications , malignant hypertension and history of atherosclerotic coronary artery disease or cerebrovascular disease .
• TT homozygotes particularly in the presence of elevated BP, develop atherosclerotic complications earlier than CC homozygotes , or CT heterozygotes .
• Table 1 -5312 C/T SNP genotype frequencies amongst the hypertensive participants in the clinical trial compared to a community dwelling control population
• Genotype CC 166 (0.640) 254 (0.66) CT 92 (0.355) 122 (0.31) TT 1 (0.004) 11 (0.03)
• Tables 2 and 3 show baseline clinic and ambulatory blood pressures, and the changes from baseline with study treatment . It is clear from these tables that BP lowering, particularly for sitting clinic pressures and night-time ambulatory BPs, was greatest with Losartan 100 mg daily amongst T allele carriers . This suggests that greater expression of renin contributes to the hypertension of T allele carriers , and through inhibition of the effects of the key end-product of the RAAS, namely angiotensin II , greater BP lowering was achieved.
• Table 4 also shows trough plasma aliskiren levels after 4 weeks of treatment . Interestingly on treatment plasma aliskiren levels were reduced by about 20% - 30% amongst T allele carriers .
• T allele carriers respond to renin inhibition and to the resultant decrease in negative feed-back by angiotensin II on the RAAS, by greatly increased expression of the renin gene by comparison to CC homozygotes .
• the excess renin produced by T allele carriers binds the drug strongly and reduces the free drug concentration.
• Haemodyamic parameter Aliskiren 37.5mg Aliskiren 75mg Aliskiren 150mg Aliskiren 300mg Losartan lOOmg (T/C 11/29) (T/C 17/25) (T/C 16/25) (T/C 15/27) (T/C 15/24)
• Table 3 Baseline ambulatory blood pressures, and changes from baseline with study treatment.
• Haemodyamic parameter Aliskiren 37.5mg Aliskiren 75mg Aliskiren 150mg Aliskiren 300mg Losartan lOOmg (T/C 11/29) (T/C 17/25) (T/C 16/25) (T/C 15/27) (T/C 15/24)
• Table 4 Trough plasma aliskiren levels and trough plasma renin activity at baseline and after four weeks of treatment.
• Plasma aliskiren on treatment (ng/ml) Ren CC homozygotes 3.1 (4.0, 1.5) 4.4 (6.4, 3.5)11.0 (16.0,6.7) 31.5 (70.3, 13.5) - Ren T allele carriers 2.4 (2.9, 1.1) 3.9 (7.3, 2.8)7.1 (12.0, 5.4) 20.5 (41.0, 17.0)
• AU available data are shown - Baseline PRA, on-treatment PRA and % change in PRA values were available for 39, 39, 40, 39 and 39 subjects in the five treatment groups
• Drug cost savings ( state or individual cost savings ) - The test will sensitively and specifically identify those patients who are likely and those who are unlikely to gain direct benefit from particular drug classes . Patients who are unlikely to benefit from a particular drug will not be prescribed unnecessary therapy.
• Frossard PM Malloy MJ. Lestringant GG . Kane JP . Haplotypes of the human renin gene associated with essential hypertension and stroke . J. Hum. Hypertens . 2001 ; 15 : 49-55.

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